There are reports that arachidonic acid, as is docosahexaenoic acid, contained in breast milk and plays a role in the infant's development (“Advances in Polyunsaturated Fatty Acid Research,” Elsevier Science Publishers, 1993, pp. 261-264) and reports on the importance of arachidonic acid in the development of fetus's height and brain (Proc. Natl. Acad. Sci. U.S.A., 90, 1073-1077 (1993), Lancet, 344, 1319-1322 (1994)), and accordingly there are moves to supplement infant formula with arachidonic acid and docosahexaenoic acid that represent major differences in the fatty acid composition between breast milk and infant formula.
The FAO/WHO has issued a recommendation that the intake of arachidonic acid and docosahexaenoic acid be 60 mg/kg/day and 40 mg/kg/day, respectively, for permature infants and the intake of arachidonic acid and docosahexaenoic acid be 40 mg/kg/day and 20 mg/kg/day, respectively, for mature infants.
As methods of obtaining these fatty acids in large amount, there are conventionally known production methods that utilize microorganisms. For example, a method utilizing a microorganism belonging to the genus Mortierella has been proposed that permits the production of arachidonic acid in a shorter culturing time, with a higher yield, and using simpler processes using an inexpensive commonly used medium (Japanese Examined Patent Publication (Kokoku) No. 7-34752).
However, the percentage of arachidonic acid in the total fatty acids is not satisfactory with these conventional production methods. Thus, when lipids containing arachidonic acid are to be added to foods, the highest possible content of arachidonic acid is preferred since it can minimize the amount of undesirable substances added and therefore it is also desirable in terms of quality and cost. It is also desirable when arachidonic acid ethyl esters are to be isolated and purified, since the highest possible content of arachidonic acid providing highly purified products with simple procedures and at low cost.
As methods of enhancing the percentage of arachidonic acid in lipids relative to the total fatty acids, there are many methods known in the art. For example, by culturing Mortierella alpina at 28° C. in a normally airation and agitation culture followed by culturing for further 6 days under the condition of complete glucose depletion, the percentage of arachidonic acid has successfully been raised to 67.4% (Appl. Microbial. Biotechnol. 31, 11-6 (1989)). This method, however, takes advantage of the fact that microorganisms in a starved state effect β-oxidation of fatty acids of triglyceride with a low degree of saturation to convert them to energy.
Therefore, there are actually no changes in the total amount of arachidonic acid, and due to the reduction of fatty acids with a low degree of saturation only the relative percentage of arachidonic acid is enhanced. Thus, it does not mean that the amount of produced triglycerides containing a high percentage of arachidonic acid is increased, but on the contrary it appears that the percentage of triglycerides is also decreased as a result of β-oxidation.
It is known that at a temperature lower than the optimum growth temperature, microorganisms capable of producing arachidonic acid generally try to adapt to low-temperature environments by enhancing the degree of unsaturation of unsaturated fatty acids so as to maintain fluidity and the functions of the cell membrane, and thereby the activity of Δ6 desaturase and Δ5 desaturase become enhanced so that fatty acids having a high degree of unsaturation such as arachidonic acid are produced in greater amounts. Culturing at a low temperature is therefore desirable to enhance the content of arachidonic acid.
In a method utilizing the above property, Mortierella alpina was cultured in an airation and agitation culture at 20° C. for 16 days in which the percentage of arachidonic acid was successfully enhanced to 71.2% (“Industrial Applications of Single Cell Oils,” American Oil Chemists' Society Champaign, 1992, pp. 52-60). It is known, however, that since this method requires a long time for culturing, it is not only unsuitable for industrial production but at the low temperature a part of arachidonic acid that was produced is converted to eicosapentaenoic acid by a ω3 desaturase (Biochem. Biophys. Res. Commun., 150, 335-341 (1988)) that acts at low temperatures thereby reducing the percentage of arachidonic acid and increasing the percentage of eicosapentaenoic acid in the total fatty acids.
For example, when filamentous fungus of the genus Mortierella is cultured at 12° C. for 7 weeks, ω3 desaturase is activated and the percentage of EPA to the total fatty acids reaches 2 to 20% (J. Am. Oil Chem. Soc., 65, 1455-1459 (1988)) and accordingly the percentage of arachidonic acid decreases. In contrast, by using strains wherein ω3 desaturase is decreased or is lacking, the percentage of arachidonic acid to the total fatty acids in the lipid can be enhanced to 50% or more, and when mutation is repeated it can be enhanced to 70% or more and the percentage of EPA can be kept at 0.5% or less.
Little eicosapentaenoic acid is contained in breast milk, and recent studies have even shown that it is detrimental to the development of babies of premature infants (“Advances in Polyunsaturated Fatty Acid Research,” Elsevier Science Publishers, 1993, pp. 261-264). Thus, there is a strong need for the development of a method that can produce lipids containing a high percentage of arachidonic acid and containing little or no eicosapentaenoic acid using an inexpensive commonly used medium, a simple process, and on a large scale.
On the other hand, dihomo-γ-linolenic acid is converted to arachidonic acid by a Δ5 desaturase irrespective of the culture temperature. As a method of producing dihomo-γ-linolenic acid on a large scale by a fermentation at low cost, there is a known method of culturing by adding a substance that inhibits the activity of Δ5 desaturase such as sesamin, episesamin, sesaminol, episesaminol, and curcumin to the medium, or a method of culturing using a mutant strain of a microorganism capable of producing arachidonic acid in which mutation has been induced so that Δ5 desaturase activity is decreased or deleted (Japanese Unexamined Patent Publication (Kokai) No. 1-243992, Japanese Unexamined Patent Publication (Kokai) No. 3-72892, Japanese Unexamined Patent Publication (Kokai) No. 3-49688, and Japanese Unexamined Patent Publication (Kokai) No. 5-91887).
In this case also, however, culturing at a temperature lower than the optimum growth temperature such as 12° C. in an attempt to enhance the dihomo-γ-linolenic acid content would result in the activation of the above-mentioned ω3 desaturase so that concern arises that a part of dihomo-γ-linolenic acid may be converted to 8, 11, 14, 17-eicosatetraenoic acid and thereby the percentage of dihomo-γ-linolenic acid may decrease and that of 8, 11, 14, 17-eicosatetraenoic acid may increase. Thus, there is a strong need for the development of a method that can produce lipids containing a high percentage of dihomo-γ-linolenic acid using an inexpensive commonly used medium, a simple process, and on a large scale.